JP2018515447A - Parthenolide and its derivatives for the treatment of axonal disorders - Google Patents

Abstract

The present invention reduces detyrosination of axonal tip microtubules selected from the group consisting of tubulin carboxypeptidase inhibitors and tubulin tyrosine ligase activators and combinations thereof for the treatment of axonal disorders It relates to the compound to be made. [Selection] Figure 4

Description

  The present invention relates to the treatment of axonal disorders during nerve injury or disease.

  The complex and delicate structures of the nervous system, ie the brain, spinal cord and peripheral nerves, are susceptible to various types of disorders. For example, injury to peripheral nerves (peripheral neuropathy) can be caused by various traumas including lacerations, local contusions (gun wounds), stretch / traction injury, compression, drug injection injury or electrical injury. In addition, peripheral neuropathy may be the result of systemic diseases such as diabetes or leprosy, vitamin deficiencies, medications such as chemotherapy, radiation therapy, alcohol overdose, immune system diseases or viral infections. CNS axonal disorders are related to diseases caused by amputation, rupture or compression / contusion, or damage to axons, such as axonal disorders and axonal destruction caused by stroke or multiple sclerosis Sometimes.

  Peripheral neuropathy is a common cause of major functional decline and medical expenditure. In the case of peripheral nerve amputation or rupture, surgery may be performed. In peripheral nerve reconstruction, damaged nerves are identified and exposed so that normal nerve tissue above and below the damage level can be observed, damaged portions of the nerves are removed, and the cut nerve endings are then carefully rejoined. However, large section injuries are not suitable for primary repair, and standard clinical management results in poor sensory and motor recovery in the majority of cases, despite the rigorous application of complex microsurgical techniques.

  In general, damaged peripheral nerve tissue is capable of regenerating severed axons and is therefore capable of repair. So-called nerve regeneration mechanisms can include the generation of new glia, axonal outgrowth, remyelination or restoration of functional synapses. However, the ability of nerve regeneration differs greatly between the peripheral nervous system (PNS) and the central nervous system (CNS). In contrast to the peripheral nervous system, central nerve axonal regeneration is very limited by the axon-inhibiting environment created by myelin-derived factors and the formation of inhibitory glial scars. Furthermore, CNS neurons have a much lower natural ability to regrow damaged axons. However, while PNS-damaged axons generally have greater intrinsic potential for axonal regrowth, functional regeneration is limited, mainly due to a decrease in neurotrophic support over time from Schwann cells and axonal guidance impairment. There are often. These aspects are particularly apparent in the case of long-distance regeneration, such as after leg sciatic nerve injury or arm median nerve injury. Therefore, it is highly desirable to develop new therapeutic methods aimed at accelerating axonal regeneration and thereby improving functional recovery.

  The object underlying the present invention was therefore to provide compounds which can be used in the treatment of axonal disorders.

  The problem is selected from the group consisting of a tubulin carboxypeptidase (TCP) inhibitor, a tubulin tyrosine ligase (TTL) activator, and combinations thereof for the treatment of axonal disorders Solved by compounds that reduce the detyrosination of axon microtubules.

  Surprisingly, it was found that the TCP inhibitor parthenolide significantly promotes axonal growth of adult dorsal root ganglion neurons in culture. Furthermore, in vivo drug administration accelerated axonal growth and promoted sciatic nerve function regeneration in wild-type mice compared to their respective controls. This could be shown with local and systemic drug administration. Thus, TCP inhibitors such as parthenolide and TTL activators provide promising drug treatments for nerve damage and improvements in nerve repair.

  The beneficial effect of parthenolide is believed to be due to a reduction in tubulin carboxypeptidase (TCP) tubulin detyrosination at the axon tip due to pharmacological inhibition of a protein designated as tubulin carboxypeptidase (TCP). As used herein, the term “tubulin carboxypeptidase inhibitor” targets TCP that cleaves -Glu-Tyr bonds and removes C-terminal tyrosine residues from native tyrosine tubulin. Refers to a group of drugs that inhibit activity or inhibit microtubule detyrosination. As used herein, the term “tubulin tyrosine ligase activator” is expressed as tubulin tyrosine ligase (TTL), which adds a COOH-terminal tyrosine residue to tubulin on the other hand. Refers to a drug that activates the activity of a target protein. The TCP inhibitor parthenolide increased axonal regeneration in cell culture.

  Of note, in vivo or intraperitoneal administration of a TCP inhibitor in vivo significantly promoted sciatic nerve regeneration and accelerated functional recovery in injured animals. Thus, a pharmacological approach to reduce axonal tip microtubule detyrosination significantly improves nerve repair and central projection repair and is a novel and clinically relevant for treating axonal disorders A good strategy. Conveniently, local and systemic administration of drugs has been shown to be effective.

  The term “axon” as used herein refers to an elongated protrusion of a peripheral or central neuron. Axons usually make electrical impulses out of the cell body of neurons. Axons and their insulating sheaths are called nerve fibers. In the central nervous system, myelin is produced by oligodendrocytes. In the peripheral nervous system, myelin is formed by Schwann cells. Peripheral nerve fibers contain axons, myelin sheaths, Schwann cells and their inner lining, and central nerve fibers do not contain Schwann cells and inner lining, but contain oligodendrocytes. As used herein, the term “axon tip” refers to the end or end of an axon. At the tip of the axon is a dynamic compartment called a growth cone, through which the axon that grows moves through its environment.

  As used herein, the term “axon disorder” refers to a disorder of any nerve fiber and nerve tissue axons. As used herein, the term “nerve” refers to sensory fibers, motor fibers, or both. The term “axonopathy” refers to nerve damage and peripheral neuropathy caused by axon disorders, as well as optic nerve or spinal cord disorders. Peripheral neuropathy may be the result of systemic diseases such as diabetes or leprosy, vitamin deficiencies, medications such as chemotherapy, radiation therapy, alcohol overdose, immune system disease or viral infection. Axonal disorders can appear during nerve injury or disease. Nerve damage can be given to nerves in the peripheral nervous system, for example by fracture or amputation of the limbs, or the spinal cord can be damaged by amputation, tearing or compression / contusion. Axonal disorders may be associated with diseases that damage the axons, such as axonal disorders and axonal destruction caused by stroke or multiple sclerosis.

  Nerve damage may be caused by trauma such as laceration, local contusion, stretch / traction injury, compression, drug injection injury or electrical injury. Specific disorders are nerve amputation, rupture or compression / contusion. Nerve damage is classified by Seddon based on whether it is continuous with the three main types of nerve fiber damage, correlating the extent of damage with symptoms, pathology and prognosis. Neulabraxia refers to a disorder of the peripheral nervous system in which axons are safe but myelin damage is present causing interference with nerve fiber impulse transmission. Axonotomesis refers to a type of injury that is the result of more severe destruction or bruise than neuraplaxia, where both nerve fibers and nerve sheaths rupture.

  Neurotomy is the most severe lesion and occurs during severe contusions, stretches or lacerations. Not only axons but also encapsulated connective tissue loses continuity. Extreme neurotomy is a truncation. In an embodiment, nerve injury refers to axonotosis or neurotomy.

  Thus, in embodiments, the axonal disorder is damage to the peripheral or central nervous system that causes neuronal loss. In a preferred embodiment, the axonal disorder is a central projection injury of the sciatic or optic nerve or spinal cord. Central projection axonopathy of the optic nerve or spinal cord is a favorable damage to the central nervous system. An additional axonal disorder of the peripheral nerve, which is preferred as well as the sciatic nerve, is a disorder of the nerve protruding into the arms and fingers. The sciatic nerve or the nerve that protrudes into the arms and fingers is a long nerve and is particularly vulnerable to injury. Disorders can lead to irreversible declines in sensory and motor function as well as pain in patients. Optic nerve damage often results in lifelong severe visual impairment, or even complete blindness of the eye. Furthermore, axonal regeneration does not normally occur in the damaged spinal cord that includes other central projections of axons. Axonal disorders can result in severe disorders such as limb paralysis or paraplegia. For this reason, the axon growth promoting effect of parthenolide can advantageously improve the clinical outcome after spinal cord injury. In other embodiments, the axonal disorder is damage to the cranial nerve, particularly the optic nerve or the trigeminal nerve, particularly its eye branch. In a further embodiment, the axonal disorder is nerve damage distributed in the brachial plexus or internal organs.

  Further disorders or diseases caused by axonal disorders can be selected from the group comprising glaucoma when axons of the optic nerve head are impaired. In embodiments, the axonal disorder is associated with peripheral neuropathy, glaucoma, stroke or multiple sclerosis. Examples of peripheral neuropathy are diabetic neuropathy and cytostatic-induced neuropathy.

  In a further embodiment, the axonal injury is related to corneal damage or denervation of the transplanted cornea, or to the lesioned optic or trigeminal nerve, in particular the axotomy fiber of the eye branch. Corneal damage can be caused by cutting or peeling. The cornea is generally the most densely innervated tissue on the body surface. However, corneal axons are cut by corneal transplantation, resulting in complete denervation of the transplanted cornea. The reinnervation of donor tissue varies widely, and sensory dullness often persists for many years after the initial surgery. Therefore, the promotion of growth from the axons originating from the optic nerve to the cornea, especially the transplanted cornea, is very advantageous.

  Since parthenolide has been shown to interact directly with the axon growth cone, it is likely that parthenolide will accelerate corneal axon growth. Conveniently, it was demonstrated that parthenolide can promote nerve regeneration not only in peripheral nerves, but also in central neurons such as retinal ganglion cells, which means that parthenolide has axotomy of the optic nerve that caused glaucoma or lesions. Indicates that it may be useful for fiber treatment.

  A preferred compound that reduces de-tyrosination of axonal tip microtubules for the treatment of axonal disorders is a tubulin carboxypeptidase inhibitor. Preferred tubulin carboxypeptidase inhibitors are parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. Parthenolide is a sesquiterpene lactone, which is usually extracted from the plant Asteraceae, also known as feverfew, Tanacetum parthenium. Parthenolide is the name for 4,5-epoxy-6α-hydroxy-gamma-lactone. The chemical formula (1) for parthenolide is shown below:

  Parthenolides contain a chiral center and can therefore be racemic, enantiomeric or stereoisomeric and can also be used. A preferred stereoisomer of parthenolide is represented by the following chemical formula (1a):

  Parthenolide follows the IUPAC nomenclature (1aR, 4E, 7aS, 10aS, 10bR) -1a, 5-dimethyl-8-methylene-2,3,6,7,7a, 8,10a, 10b-octahydroxy oxyreno Also represented as [9,10] cyclodeca [1,2-b] furan-9 (1aH) -one. It was shown that parthenolide can significantly promote neuronal axon growth in vitro, can promote peripheral nerve regeneration (sciatic nerve), and can significantly accelerate functional recovery after injury in vivo. In addition, parthenolide promotes neurite outgrowth of CNS neurons such as cultured retinal ganglion cells.

  In embodiments, parthenolide derivatives or structural analogs may also be used for TCP inhibition. In embodiments, the parthenolide derivative comprises 8-, 9-, or 14-hydroxypartenolide and / or 13-aminopartenolide, such as 13-dimethylaminopartenolide commonly referred to as dimethylaminopartenolide (DMAPT). Selected from the group. Dimethylaminopartenolide (DMAPT) is advantageously a water-soluble orally bioavailable parthenolide derivative.

  Such parthenolide derivatives can increase the solubility of the compound, which may be advantageous for formulating appropriate pharmaceutical formulations, providing improved bioavailability of the compound in an aqueous environment To do. The hydroxy derivative may be selected from the group comprising 8-, 9- or 14-hydroxypartenolide, especially hydroxy-8a-partenolide. The 13-aminopartenolide derivatives include 11βH, 13-dimethylaminopartenolide, 11βH, 13-diethylaminopartenolide 11βH, 13- (tert-butylamino) partenolide, 11βH, 13- (pyrrolidin-1-yl) partenolide, 11βH, 3- (Piperidin-1-yl) partenolide, 11βH, 13- (morpholin-1-yl) partenolide, 11βH, 13- (4-methylpiperidin-1-yl) partenolide, 11βH, 13- (4-methylpiperazine-1- Yl) partenolide, 11βH, 13- (homopiperidin-1-yl) partenolide, 11βH, 13- (heptamethyleneimine-1-yl) partenolide, 11βH, 13- (azetidin-1-yl) partenolide and / or The 11BetaH, may be selected from the group comprising 13-diallyl-amino parthenolide. A preferred aminopartenolide is 13-dimethylaminopartenolide.

  Parthenolide structural analogs can also function as TCP inhibitors. In embodiments, the structural analog of parthenolide is kunicin, compounds of formula (2) and (3) below, and / or racemates, enantiomers, stereoisomers, solvates, hydrates, and Selected from the group comprising pharmaceutically acceptable salts and / or esters:

  Knissin follows the IUPAC nomenclature [(1R, 2S, 4E, 8Z, 10S) -8- (hydroxymethyl) 4-methyl-13-methylidene-12-oxo-11-oxabicyclo [8.3.0] Trideca-4,8-dien-2-yl] (3S) -3,4-dihydroxy-2-methylidene-butanoate or [(3aR, 4S, 6E, 10Z, 11aR) -10- (hydroxymethyl)- 6-Methyl-3-methylidene-2-oxo-3a, 4,5,8,9,11a-hexahydrocyclodeca [b] furan-4-yl] (3R) -3,4-dihydroxy-2-methyl Also referred to as redenbutanoate. A preferred stereoisomer of kunisin is represented by the following chemical formula (4):

  Parthenolide may be further available in the form of its solvates, hydrates, and pharmaceutically acceptable salts and esters. The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. Pharmaceutically acceptable salts can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases, organic anions, organic cations, halides or alkalis. The term pharmaceutically acceptable salts includes alkali metal salts and addition salts of free acids or free bases. Suitable pharmaceutically acceptable base addition salts of the fusion protein include metal salts and organic salts. Preferred salts derived from inorganic bases include ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amine salts. Parthenolide and its derivatives or analogs can be used in the form of hydrochloride or maleate.

  Especially for intra-nerve administration, the concentration of the compound, in particular parthenolide, is in the range ≧ 0.01 μM to ≦ 10 μM, in particular ≧ 0.01 μM to ≦ 1 μM, or ≧ 0.05 μM to ≦ 0.1 μM, or ≧ 0.05 μM. It can be in the range of ~ ≦ 1 μM.

  Compounds that reduce microtubule detyrosination, such as parthenolide, can be used alone or in combination with other therapeutic ingredients in the treatment of axonal disorders. In embodiments, a compound that reduces microtubule detyrosination, such as parthenolide as described above, can be used in combination with a compound selected from ROCK inhibitors such as paclitaxel, epothilone B and / or Y27632. Paclitaxel follows the IUPAC nomenclature (2α, 4α, 5β, 7β, 10β, 13α) -4,10-bis (acetyloxy) -13-{[(2R, 3S) -3- (benzoylamino) -2 -Hydroxy-3-phenylpropanoyl] oxy} -1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate, available under the trade name Taxol . The combination with paclitaxel or epothilone B may be particularly advantageous for the treatment of central nervous system axonal disorders. As used herein, the term “ROCK inhibitor” refers to a drug that targets a protein designated as Rho-related protein kinase (ROCK) and inhibits its activity. Suitable ROCK inhibitors are the compounds represented by Y27632 or (1R, 4r) -4-((R) -1-aminoethyl) -N- (pyridin-4-yl) cyclohexanecarboxamide dihydrochloride.

  Compounds that reduce axonal tip microtubule detyrosination may be formulated as pharmaceutical compositions. A further aspect of the invention is the detyrosination of microtubules at the tip of axons selected from the group consisting of tubulin carboxypeptidase inhibitors and tubulin tyrosine ligase activators and combinations thereof for the treatment of axonal disorders The present invention relates to a pharmaceutical composition comprising as an active ingredient a compound that reduces sucrose.

  Preferred compounds are TCP inhibitors. In pharmaceutical composition embodiments, the TCP inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structure. It is analog. In embodiments, the parthenolide derivative is selected from the group comprising 13-aminopartenolide, such as 8-, 9- or 14-hydroxypartenolide and / or 13-dimethylaminopartenolide (DMAPT). The hydroxy derivative may be selected from the group comprising 8-, 9- or 14-hydroxypartenolide, especially hydroxy-8a-partenolide. The 13-aminopartenolide derivatives include 11βH, 13-dimethylaminopartenolide, 11βH, 13-diethylaminopartenolide 11βH, 13- (tert-butylamino) partenolide, 11βH, 13- (pyrrolidin-1-yl) partenolide, 11βH, 3- (Piperidin-1-yl) partenolide, 11βH, 13- (morpholin-1-yl) partenolide, 11βH, 13- (4-methylpiperidin-1-yl) partenolide, 11βH, 13- (4-methylpiperazine-1- Yl) partenolide, 11βH, 13- (homopiperidin-1-yl) partenolide, 11βH, 13- (heptamethyleneimine-1-yl) partenolide, 11βH, 13- (azetidin-1-yl) partenolide and / or The 11BetaH, may be selected from the group comprising 13-diallyl-amino parthenolide. A preferred aminopartenolide is 13-dimethylaminopartenolide. In further embodiments, the structural analogs of parthenolide are kunicin, compounds of formula (2) and (3), and / or racemates, enantiomers, stereoisomers, solvates, hydrates, and pharmaceuticals thereof Selected from the group comprising acceptable salts and / or esters.

  In embodiments, the pharmaceutical composition is for the treatment of axonal disorders of the peripheral or central nervous system, particularly central projection damage of the sciatic or optic nerve or spinal cord. In embodiments, the pharmaceutical composition is for the treatment of axonal disorders associated with peripheral neuropathy, glaucoma, stroke or multiple sclerosis. Examples of peripheral neuropathy are diabetic neuropathy and cytostatic neuropathy. In a further embodiment, the pharmaceutical composition comprises an axonal disorder associated with denervation of an injured or transplanted cornea, or an axon associated with a lesioned optic or trigeminal nerve, in particular an axotomy fiber of the eye branch For the treatment of faults.

  The pharmaceutical composition may comprise a compound that reduces axonal tip microtubule detyrosination according to the invention as an active ingredient, a pharmaceutically acceptable carrier and optionally other therapeutic ingredients or adjuvants. Accordingly, the present invention also relates to a pharmaceutical composition for the treatment of axonal disorders, said composition comprising as an active ingredient a compound that reduces detyrosination of axonal tip microtubules according to the present invention, and pharmaceutically Contains an acceptable carrier.

  The compound can be dissolved or dispersed in a pharmaceutically acceptable carrier. The term “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions, or other adverse reactions when administered to a subject, such as a human, as appropriate. Say. The drug carrier can be, for example, a solid, liquid, or gas. Suitable carriers and adjuvants can be solid or liquid and correspond to materials commonly used in pharmaceutical technology for pharmaceutical formulations. For example, water, glycols, oils, alcohols, etc. can be used to form liquid formulations such as solutions. Examples of solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

  The composition may be suitable for oral or parenteral administration. Parenteral administration includes subcutaneous administration, intramuscular administration, intravenous administration, intraneural administration, perineural administration, intraperitoneal administration, and topical administration. In embodiments, the composition is formulated for topical administration, such as intraneural or perineural administration, or for systemic administration, such as intraperitoneal, intravenous, subcutaneous, or oral administration. For the treatment of peripheral nerves, the composition can be administered systemically or intranerveally, for example, into the sciatic nerve. Also, local or local administration such as perineural administration may be preferred. In further embodiments, the composition is formulated for intraocular administration or as eye drops. Administration in the form of eye drops may be particularly useful in the treatment of damaged or transplanted cornea or glaucoma, or lesioned optic or trigeminal nerves, especially axotomy fibers of the eye branch. Compositions suitable for injection include sterile aqueous solutions or dispersions.

  Especially for intra-nerve administration, the concentration of the compound, in particular parthenolide, is in the range ≧ 0.01 μM to ≦ 10 μM, in particular ≧ 0.01 μM to ≦ 1 μM, or ≧ 0.05 μM to ≦ 0.1 μM, or ≧ 0.05 μM. It can be in the range of ~ ≦ 1 μM. Already concentrations in the range of 0.01 μM to 0.1 μM significantly increase axonal regeneration after sciatic nerve injury in vivo, while higher concentrations above 10 μM rather decrease axonal regeneration I understood.

  For example, in the treatment of the central nervous system, the composition may be administered locally, for example by nerve injection, minipumps, etc., but it is preferred that the composition is administered via the gastrointestinal route. In particular, the pharmaceutical composition may be administered systemically, for example orally, systemically or intraperitoneally. In further embodiments, the composition may be administered as eye drops. Intraocular administration in the form of eye drops may be particularly useful in the treatment of damaged or transplanted cornea or glaucoma, or lesioned optic or trigeminal nerves, especially axotomy fibers of the eye branch . The pharmaceutical compositions are conveniently provided in unit dosage form and can be prepared by any of the methods known in the art of dispensing. The pharmaceutical compositions can be produced under aseptic conditions using standard pharmaceutical techniques well known to those skilled in the art.

  The pharmaceutical composition may include a compound that reduces microtubule detyrosination, such as parthenolide, alone or in combination with other therapeutic ingredients. Preferred therapeutic ingredients provide an anti-inhibitory effect on central myelin or glial scar inhibitors. In embodiments, the pharmaceutical composition activates a combination of a compound that reduces microtubule detyrosination, such as parthenolide as described above, and a compound selected from ROCK inhibitors, such as paclitaxel, epothilone B and / or Y27632. Contains as an ingredient. A pharmaceutical composition comprising paclitaxel or epothilone B as an additional therapeutic component may be particularly useful for the treatment of central nervous system axonal disorders.

  The present invention relates to an axonal tip microtubule selected from the group consisting of a tubulin carboxypeptidase inhibitor and a tubulin tyrosine ligase activator and combinations thereof for the manufacture of a medicament for the treatment of axonal disorders. It also relates to the use of compounds that reduce the detyrosination of. In embodiments, the TCP inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. . In embodiments, the parthenolide derivative is selected from the group comprising 13-aminopartenolide, such as 8-, 9- or 14-hydroxypartenolide and / or 13-dimethylaminopartenolide (DMAPT). The hydroxy derivative may be selected from the group comprising 8-, 9- or 14-hydroxypartenolide, especially hydroxy-8a-partenolide. The 13-aminopartenolide derivatives include 11βH, 13-dimethylaminopartenolide, 11βH, 13-diethylaminopartenolide 11βH, 13- (tert-butylamino) partenolide, 11βH, 13- (pyrrolidin-1-yl) partenolide, 11βH, 3- (Piperidin-1-yl) partenolide, 11βH, 13- (morpholin-1-yl) partenolide, 11βH, 13- (4-methylpiperidin-1-yl) partenolide, 11βH, 13- (4-methylpiperazine-1- Yl) partenolide, 11βH, 13- (homopiperidin-1-yl) partenolide, 11βH, 13- (heptamethyleneimine-1-yl) partenolide, 11βH, 13- (azetidin-1-yl) partenolide and / or The 11BetaH, may be selected from the group comprising 13-diallyl-amino parthenolide. A preferred aminopartenolide is 13-dimethylaminopartenolide. In further embodiments, the structural analogs of parthenolide are kunicin, compounds of formula (2) and (3), and / or racemates, enantiomers, stereoisomers, solvates, hydrates, and pharmaceuticals thereof Selected from the group comprising acceptable salts and / or esters. In embodiments, the axonal disorder is damage to the peripheral or central nervous system, particularly central projection of the sciatic or optic nerve or spinal cord. In embodiments, the axonal disorder is associated with peripheral neuropathy, glaucoma, stroke or multiple sclerosis. Examples of peripheral neuropathy are diabetic neuropathy and cytostatic neuropathy. In a further embodiment, the axonal disorder is associated with denervation of the damaged or transplanted cornea, or with the lesioned optic nerve or trigeminal nerve, in particular the axotomy fibers of the eye branch.

  Compounds that reduce microtubule detyrosination, such as parthenolide, can be used alone or in combination with other therapeutic agents. In embodiments, the use is for the manufacture of a medicament for the treatment of axon disorders, compounds that reduce microtubule detyrosination, such as parthenolide as described above, and ROCK inhibition, such as paclitaxel, epothilone B and / or Y27632. Relates to the use of compounds selected from agents.

  A further aspect of the present invention relates to a method of treating an axonal disorder, wherein the method comprises a neurite tip microbe selected from the group consisting of a tubulin carboxypeptidase inhibitor and a tubulin tyrosine ligase activator and combinations thereof. Administering to the subject a therapeutically effective amount of a compound that reduces vascular detyrosination.

  Subjects include both human and animal subjects, particularly medical human subjects or mammalian subjects such as mice or rats. The term “therapeutically effective amount” is used herein to mean an amount or dose sufficient to cause a clinically significant improvement in a condition in a subject.

  In embodiments, the method refers to administering a TCP inhibitor. In embodiments, the TCP inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. . In embodiments, the parthenolide derivative is selected from the group comprising 13-aminopartenolide, such as 8-, 9- or 14-hydroxypartenolide and / or 13-dimethylaminopartenolide (DMAPT). The hydroxy derivative may be selected from the group comprising 8-, 9- or 14-hydroxypartenolide, especially hydroxy-8a-partenolide. The 13-aminopartenolide derivatives include 11βH, 13-dimethylaminopartenolide, 11βH, 13-diethylaminopartenolide 11βH, 13- (tert-butylamino) partenolide, 11βH, 13- (pyrrolidin-1-yl) partenolide, 11βH, 3- (Piperidin-1-yl) partenolide, 11βH, 13- (morpholin-1-yl) partenolide, 11βH, 13- (4-methylpiperidin-1-yl) partenolide, 11βH, 13- (4-methylpiperazine-1- Yl) partenolide, 11βH, 13- (homopiperidin-1-yl) partenolide, 11βH, 13- (heptamethyleneimine-1-yl) partenolide, 11βH, 13- (azetidin-1-yl) partenolide and / or The 11BetaH, may be selected from the group comprising 13-diallyl-amino parthenolide. Parthenolide structural analogs can also function as TCP inhibitors. A preferred aminopartenolide is 13-dimethylaminopartenolide. In further embodiments, the structural analogs of parthenolide are kunicin, compounds of formula (2) and (3), and / or racemates, enantiomers, stereoisomers, solvates, hydrates, and pharmaceuticals thereof Selected from the group comprising acceptable salts and / or esters.

  In embodiments, the methods relate to administering a compound selected from a microtubule detyrosination such as the above-mentioned parthenolide and a ROCK inhibitor such as paclitaxel, epothilone B and / or Y27632.

  In embodiments, the axonal disorder is damage to the peripheral or central nervous system, particularly central projection of the sciatic or optic nerve or spinal cord. In embodiments, the axonal disorder is associated with peripheral neuropathy, glaucoma, stroke or multiple sclerosis. Examples of peripheral neuropathy are diabetic neuropathy and cytostatic neuropathy. In a further embodiment, the axonal disorder is associated with denervation of the damaged or transplanted cornea, or with the lesioned optic nerve or trigeminal nerve, in particular the axotomy fibers of the eye branch.

  Treatment can include oral or parenteral administration. Parenteral administration includes subcutaneous administration, intramuscular administration, intravenous administration, intraneural administration, perineural administration, intraperitoneal administration, and intraperitoneal administration. In embodiments, the composition is administered via an intraneuronal route, a perineural route, an intraperitoneal route, an intravenous route, a subcutaneous route, or an oral route. For the treatment of peripheral nerves, the composition can be administered intraneurally, for example, into the sciatic nerve. Also, topical parthenolide administration such as perineural administration may be preferred. Especially for intra-nerve administration, the concentration of the compound, in particular parthenolide, is in the range ≧ 0.01 μM to ≦ 10 μM, in particular ≧ 0.01 μM to ≦ 1 μM, or ≧ 0.05 μM to ≦ 0.1 μM, or ≧ 0.05 μM. It can be in the range of ~ ≦ 1 μM. In the treatment of the central nervous system, for example, it may be preferable to administer the composition via the digestive tract route. For the treatment of damaged corneal tissue or transplanted cornea or glaucoma, it may be preferable to administer the composition as an eye drop.

  The treatment may be a continuous long-term treatment or may include a single or several administrations. Already a single injection has been found to be sufficient to accelerate sciatic nerve functional regeneration in vivo compared to solvent-treated controls.

  Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

  The following examples serve to illustrate the invention in more detail, but without limiting it.

FIG. 1A is a diagram of DRG neurons dissociated from wt mice treated with the various parthenolide concentrations shown and stained for βIII-tubulin after 2 days of culture. Scale bar: 100 μm. FIG. 1B shows the quantification of axon growth in neuronal cultures. Error bars represent the standard error of the mean value. Statistical significance was determined by ANOVA and post horm Sidac test. Treatment effect compared to solvent-treated control group: ^*** p ≦ 0.001. FIG. 1C shows dissociation from wt or GSK3 ^{S / A} double knock-in mice (α / β), solvent (−), 5 μM GSK3 inhibitor SB216673 (sb), 1 nM parthenolide (par), 5 μM sb and 1 nM FIG. 5 shows quantification of axon growth of dorsal root ganglion (DRG) neurons after 2 days of culture treated with 10 nM nocodazole (noco) or par + noco combination. Error bars represent the standard error of the mean value. Statistical significance was determined by ANOVA and post horm Sidac test. Treatment effect was compared with the vehicle-treated wt ^{group: *** p ≦ 0.001; ** p} ≦ 0.01. Treatment effect compared to vehicle-treated alpha / beta group ^{### p ≦ 0.001; ## p ≦} 0.01. FIG. 1D is a culture diagram showing βIII-tubulin positive axons of adult DRG neurons growing in microchannels of a two compartment chamber one day after axotomy. As shown, vehicle (veh) or 5 nM parthenolide was administered into the cell body compartment (par, soma) or axon compartment (par, axon). Scale bar: 250 μM. FIG. 1E shows the quantification of axonal growth of cultures in microchannels in a two compartment chamber one day after axotomy. Data from five experiments were averaged. Error bars represent the standard error of the mean value. Statistical significance was determined by ANOVA and post horm Sidac test. Treatment effect: ^** p ≦ 0.01. Non-deprivation of cultures from wild type (wt) and GSK3S / A double knock-in mice (α / β) 3 days after exposure to vehicle (−), 5 μM SB216763 (sb) or 10 nM parthenolide (par) It is the figure which showed fixed_quantity | quantitative_assay of the tyrosine tubulin positive axon tip. Data from three independent experiments were normalized to the solvent treated wt group. Error bars represent the standard error of the mean value. Statistical significance was determined by ANOVA and post horm Sidac test. Treatment effect: ^*** p ≦ 0.001. FIG. 5 shows quantification of axonal regeneration into the sciatic nerve 3 days after sciatic nerve crush, based on SCG10 stained axons after treatment with vehicle (veh) or various parthenolide concentrations in the figure. The number of animals per treatment group is illustrated. FIGS. 4A and 4B show longitudinal sections of the sciatic nerve 3 days after SNC injected intranervely with vehicle (veh) or 50 nM parthenolide (par). Regenerating axons were immunohistochemically stained for SCG10 (FIG. 4A) or pMAP1B (FIG. 4B). Scale bar: 500 μm. The asterisk indicates the extinction site. FIG. 4C shows axes of animals treated with vehicle (veh) or parthenolide as described in A / B at>1.5,>2,> 2.5 and> 3 millimeters from the site of sciatic nerve injury. It is the figure which showed fixed_quantity | quantitative_assay of a cord. Error bars represent the standard error of the mean value. Statistical significance was determined by ANOVA and post horm Sidac test. Treatment effect: ^*** p ≦ 0.001. FIG. 4D shows α-bungarotoxin (BTX) and neurofilament (BTX) of whole thumb extensor extensor tissue specimens of mice treated with parthenolide (par) or vehicle (veh) 4 days after sciatic nerve crush. It is the figure which showed NF: neurofilament (stain). White arrows indicate the reconstructed synapses of mice treated with parthenolide. Scale bar: 50 μM. FIG. 4E shows adult wild type treated with parthenolide (par, n = 11) or vehicle (veh, n = 11) 1, 4, 7, 9, 12, 14 and 21 days after sciatic nerve crush (dpc). It is the figure which showed the fixed_quantity | quantitative_assay of the motor function recovery | decision determined by static scastic index (SSI) in a mouse | mouth. ^** p ≦ 0.01; ^*** p ≦ 0.001. FIG. 4F shows the von Frey test in adult wild type mice treated with parthenolide (par, n = 11) or vehicle (veh, n = 11) 1, 4, 7, 12, 14 and 21 days after sciatic nerve crush. It is the figure which showed the fixed_quantity | quantitative_assay of the sensory function recovery | decision determined by this. Treatment effect: ^** p ≦ 0.01; ^* p ≦ 0.05. FIG. 6 shows quantification of axonal growth in wt DRG cultures after treatment with various kunisin and parthenolide (par) concentrations. Treated neuron data were normalized to vehicle controls with mean axon length of 933 μm / neuron for parthenolide and 546 μm / neuron for kunicin. Data represent the mean ± SEM of three independent experiments. Treatment effects compared to solvent control: ^** p ≦ 0.01, ^*** p ≦ 0.001, ## p ≦ 0.01, ## p ≦ 0.001. FIG. 6A) shows a single intraneuronal (in) injection of sciatic nerve crush (SNC) and vehicle (top) or parthenolide (6.25 pg par; medium) or intraperitoneal (ip) of parthenolide. It is the figure which showed the representative longitudinal section of the sciatic nerve 3 days after performing any of injection | pouring (200 ng / kg; lower). Regenerated axons were immunohistochemically stained for SCG10. Scale bar: 500 μm. The asterisk indicates the extinction site. 6B to 6D are enlarged views of each region shown in FIG. Figures 6E, F) show 1.5, 2 and 2 from the site of sciatic nerve injury in mice injected intrasolventally (E) or intraperitoneally (F) with vehicle (veh) or various doses of parthenolide (par). FIG. 5 shows axon quantification at 2.5, 3 and 3 millimeters ahead. Data represent the mean ± SEM of 5 fragments from at least 6 individual mice per experimental group. Treatment effects compared to vehicle-injected animals: ^* p ≦ 0.05, ^** p ≦ 0.01, ^*** p ≦ 0.001. FIG. 5 shows quantification of neurite outgrowth of adult retinal ganglion cells after treatment with 1 nM parthenolide (par), lithium (li) or both. Treatment effect compared to solvent control ('): ^** p ≦ 0.01.

Materials and methods:
Parthenolide was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in dimethyl sulfoxide (DMSO) to obtain the appropriate concentration. Nocodazole was purchased from Sigma-Aldrich and dissolved in DMSO to obtain the appropriate concentration. SB216673 was purchased from Sigma-Aldrich and dissolved in DMSO to obtain the appropriate concentration.

Surgical procedures:
Adult male and female (8-12 weeks) wild type and GSK3α ^S21A / GSK3β ^S9A mice (Prof. Dr. Alessi (Prof. Dr. Alessi), University of Dundee) with diet background of C57BL / 6,129 / Ola genetic background It was maintained on a 12 hour light / dark cycle as a free ingestion and used in all studies except experiments to examine the effects of parthenolide. Parthenolide experiments were performed with C57BL / 6J background mice. Animals were housed for at least 10 days under the same conditions prior to use in the experiment. Sciatic nerve crush (SNC) was performed as described in Gobrecht et al., Nature Communications. 2014; 5: 4561. Briefly, animals are anesthetized by intraperitoneal injection of ketamine (60-80 mg / kg, Pfizer, New York, NY, USA) and xylazine (10-15 mg / kg, Bayer, Leverkusen, Germany). did. A skin incision of about 10 mm was made in the buttocks, and the right sciatic nerve was exposed from the sciatic notch to the branching of the three branches. The posterior muscle tissue of the lower limbs was carefully spread while minimizing tissue damage. A Dumon # 5 forceps (Hermle, Tuttlingen, Germany) was used to crush injuries for 30 seconds proximal to the tibial bifurcation and marked with carbon (Sigma). The skin was closed using 6-0 suture.

  Immediately following nerve crush, a microcapillary and Nanoject II ™ injector (Drummond Scientific, Bloomall, Pa., USA) were used to inject the site of the sciatic nerve injury. Each 69 nl injection was performed 5 times in succession at a rate of 23 nl / sec and an interval of 30 sec.

DRG neuron culture and immunocytochemical staining procedures:
Isolate dorsal root ganglion (DRG) neurons from adult wild type (wt) and GSK3α / GSK3P mice as described in Gobrecht et al., Nature Communications 2014; 5: 4561. did. DRG (T8-L6) was collected and 0.25% trypsin / EDTA (GE Healthcare, Chalfont St Giles, UK) in DMEM (Life Technologies, Carlsbad, CA) And 0.3% collagenase type IA (Sigma, St. Louis, MO), incubated for 45 minutes at 37 ° C. and 5% CO ₂ and mechanically dissociated. 10% fetal bovine serum (GE Healthcare), penicillin / streptomycin (500 U / ml; Merck Millipore, Billerica, Massachusetts, USA) and 5-fluoro-2'-desoxyuridine (100 nM; Sigma Cells were resuspended in DMEM containing (Sigma)). In a 96-well plate (Nunc, Germany) coated with poly-D-lysine (PDL, 0.1 mg / ml; molecular weight <300,000 kDa; Sigma) and laminin (20 μg / ml; Sigma), Cells were cultured at 37 ° C. and 5% CO ₂ .

  To assess the level of MAP1B phosphorylation at threonine 1265 or detyrosinized tubulin at the tip of neuronal axons, cell cultures were treated with vehicle, 5 μM SB216673 or 1 nM parthenolide and cultured for 3 days. Ββ-tubulin (1: 2,000; Covance) and pMAP1B (1: 1000; Thermo Scientific) or detyrosinated tubulin (1: 2000; Millipore) Cultures were stained with antibodies against. The axon tip was considered to be the last 15 μm of the βIII-tubulin positive neuron extension. Data are presented as mean ± SEM of three replicate wells from at least two separate experiments. Significance of differences between groups was assessed using the Holm-Sidak post hoc test after one-way or two-way analysis of variance (ANOVA).

Two compartment chamber culture:
A two-compartment chamber (AXIS ™ axon separation device, Millipore) was placed in a culture dish coated with PDL and laminin. Adult mouse DRG neurons were cultured for 3 days in the cell body compartment until the neurons extended axons into all microchannels.

  The neuronal axons were then severed by quickly removing the medium from the axon compartment.

  Thereafter, parthenolide (5 nM) or a solvent was administered to the cell body side or axon side. A hydrostatic pressure difference was established between the cell body chamber and the axon chamber to produce a fluid flow that prevents diffusion of the drug into the microchannel.

  After further incubation for 24 hours, the neurons were fixed with 4% PFA. Mean axon length was quantified after immunocytochemical βIII-tubulin staining.

α-bungarotoxin (BTX) long thumb extensor muscle staining:
To analyze neuromuscular junction reconstruction, mice were sacrificed 4 days after SNC. The long extensor extensors were dissected and postfixed with PFA for 1 hour as described by Gobrecht et al., Nature Communications 2014; 5: 4561. The muscle was then permeabilized with 2% Triton X in PBS overnight. Axons were labeled with antibodies against neurofilament (1: 2,000; Abcam). Synapses were visualized by incubating with Alexa594-bound α-bungarotoxin (BTX) (1: 1,000; Invitrogen) in PBS-T for 1 hour.

Quantification of regenerative axons of sciatic nerve tissue:
The sciatic nerve was isolated, postfixed for 6 hours, transferred to 30% sucrose overnight at 4 ° C., and embedded in Tissue-Tek (Sakura, Leiden, The Netherlands). Longitudinal and transverse sections are cut with a cryostat (Leica, Wetzlar, Germany) and thawed on coated glass slides (Superfrost plus, Fisher, Pittsburgh, Pa., USA) for future use Stored at -20 ° C.

  A frozen section (14 μm) of the sciatic nerve was taken against the regeneration-related protein SCG10 (1: 1,500; Novus Biologicals), Cambridge, UK) or pMAP1B (1: 500; Thermo Scientific (Thermo Scientific) Immunohistochemical staining with antibodies against Scientific)). SCG10 positive axons were quantified at various points beyond the carbon-labeled lesion site as described by Gobrecht et al., Nature Communications 2014; 5: 4561. After one-way ANOVA, the Holm-Sidak post hoc test was used to assess the statistical significance of the difference between groups. Each experimental group contained at least 5 sections from 5 mice.

Static scitic index:
Eleven C57BL / 6Js injected with solvent intranerveally as described in Baptista AF et al., Journal of Neuroscience Methods. 2007; 161 (2): 259-64. Motor function recovery was quantified in mice and 11 animals treated with parthenolide by calculating a static sciatic index (SSI).

  The mouse was lifted from the ground and the left and right hind legs were photographed respectively. Opening of contralateral (C, left) and ipsilateral (I, right) footpads after sciatic nerve crush, wild type 0, 1, 4, 7, 9, 11, 14, 17 and 21 days after SNC And in transgenic GSK3 knock-in mice, it was evaluated by measuring the foot length (PL) and the distance between the first and fifth toes (FF). As described in Baptista AF et al., Journal of Neuroscience Methods. 2007; 161 (2): 259-64, static scitic index SSI can be calculated based on the already described formula. Calculated: SSI = 101.3 ((IFF-CFF) / CFF) −54.03 ((IPL−CPL) / CPL) −9.5.

  Data are expressed as mean and ± SEM of 8-10 animals per experimental group.

  After two-way ANOVA, the Holm-Sidak post hoc test was used to assess the statistical significance of the difference between groups.

Von Free test:
As described in Gobrecht et al., Nature Communications 2014; 5: 4561, von Frey filament test in 11 animals per experimental group from 0, 1, 4, 7, 14 and 11 After 21 days, recovery of sensory function after SNC was determined. The test was performed by the same experimenter at the same time of day.

  For this purpose, mice were placed on an elevated metal grid (grid size: 2 mm) and allowed to acclimate for 15 minutes before testing. Later on the ipsilateral to a series of innocuous von Frey filaments (Muromachi Kikai Co., Ltd., Tokyo, Japan) by starting with the smallest filament and increasing the filament size until a positive response is indicated by a quick escape of the foot The foot reaction was examined. After two-way ANOVA, the Holm-Sidak post hoc test was used to assess the statistical significance of the difference between groups.

Axonal regeneration by inhibition of tubulin carboxypeptidase at the tip of axon by in vitro parthenolide 1.1 Determination of axon growth in culture DRG neurons dissociated from adult wild-type mice as described above against axonal growth of mature neurons The effect of the tubulin carboxypeptidase (TCP) inhibitor parthenolide was determined. Cells were treated with vehicle, 0.1 nM, 1 nM, 2.5 nM, 5 nM, 10 nM or 100 nM parthenolide and cultured for 2 days.

  FIG. 1A shows cells treated with solvent, 1 nM, 10 nM and 100 nM parthenolide and stained for βIII-tubulin after 2 days of culture. FIG. 1B shows the quantification of axon growth in neuronal cultures. Data from three independent experiments were normalized to a vehicle-treated control group with an average axon length of 933 μm / neuron. As can be seen from FIGS. 1A and 1B, parthenolide increased axon growth significantly and concentration-dependently. The strongest effect was measured from 1 nM to 5 nM, but concentrations above 100 nM reduced axonal growth in culture. This indicates that the effect of parthenolide was concentration dependent.

1.2 Determination of microtubule dynamic regulation To verify that parthenolide-induced axonal growth was mediated by microtubule dynamic regulation, microtubule destabilization and axonal growth could be reduced. The effect of parthenolide was evaluated against the known glycogen synthase kinase 3 (GSK3: Glycogen synthase kinase 3) inhibitors SB216673 (sb) and nocodazole (noco).

Glycogen synthase kinase 3 (GSK3) is a protein kinase containing two isoforms (GSK3α and GSK3β). Both isoforms are phosphorylated and inactivated via phosphatidylinostide 3-kinase (PI3K) / AKT signaling during sciatic nerve crush (SNC). In GSK3α / GSK3β double knock-in mice (GSK3 ^{S / A} ) in which serine 21 of GSK3α and serine 9 of GSK3β are substituted with alanine, inhibitory GSK3 phosphorylation by AKT is prevented, thereby constitutively activating GSK3. The Maintained GSK3 activity significantly accelerates axonal regeneration after sciatic nerve crush. These effects are associated with increased phosphorylation of MAP1B.

DRG neurons dissociated from adult wild-type mice and GSK3α / GSK3β double knock-in mice (α / β) were respectively solvent (veh), 1 nM parthenolide, 5 μM GSK3 inhibitor SB216673 (sb), 1 nM parthenolide (par), Treated with a combination of 5 μM sb and 1 nM par, 10 nΜ nocodazole (noco), or par + noco and cultured for 2 days. FIG. 1C shows the quantification of each axonal growth after 2 days of culture. As can be seen from FIG. 1C, SB216673 did not affect the beneficial effects of parthenolide, but effectively blocked axon growth promoted by GSK3α ^{S / A} / GSK3β ^{S / A.} In contrast, nocodazole inhibits the beneficial effects of GSK3α ^{S / A} / GSK3β ^{S / A} and parthenolide to the same extent, and parthenolide and GSK3α ^{S / A} / GSK3β ^{S / A} may increase sensitivity to microtubule disrupting agents. It was suggested.

  This finding indicates that the positive effect of parthenolide on axonal regeneration is likely mediated by modulation of microtubule dynamics.

1.3 Determination of the interaction of parthenolide with existing axons Parthenolide interacted directly with existing axons rather than initiating axon formation in culture, and the axis of DRG neurons that had already been stimulated for growth In order to verify that it is sufficient to promote cord regeneration, a two-compartment culture platform was utilized that allowed fluid separation between the cell body compartment and the axon compartment as described above. For this purpose, adult DRG neurons were cultured in a two-compartment chamber for 3 days to regenerate and then axons were cut. One day after axotomy, vehicle (veh) or 5 nM parthenolide is administered either in the cell body compartment (par, soma) or in the axon compartment (par, axon), and the other compartment is filled with solvent, respectively. Administered. Solvent was administered to each compartment of the control cells.

  FIG. 1D shows βIII-tubulin positive axons of adult DRG neurons growing in microchannels of a two compartment chamber. FIG. 1E shows quantification of axonal growth one day after axotomy. Data from five experiments were averaged. As can be seen in FIG. 1E, cell body chamber parthenolide did not increase axonal growth above the level of solvent treatment, whereas axon chamber parthenolide significantly increased axonal regeneration. This indicates that the length of existing axons has been increased by parthenolide.

  These observations indicate that moderate pharmacological TCP inhibition by parthenolide increases microtubule dynamics, leading to enhanced axonal growth in cultured neurons.

Determination of Parthenolide Induced Microtubule Detyrosination In Vitro To verify that the axonal growth of mature neurons induced by parthenolide was promoted through inhibition of microtubule detyrosination. Axonal tips of cultured DRG neurons from type (wt) or GSK3S / A double knock-in mice (α / β) were added to vehicle (−) or 5 μM GSK3 inhibitor SB216673 (sb) or 10 nM parthenolide (par) Exposed to. Three days after exposure, axons were stained for detyrosinated tubulin and βIII-tubulin. Parthenolide treatment was found to reduce the level of detyrosinated tubulin at the axon tip. FIG. 2 shows non-desorption of cultures from wt and GSK3S / A double knock-in mice (α / β) 3 days after exposure to vehicle (−), 5 μM SB216673 (sb) or 10 nM parthenolide (par). It is the figure which showed fixed_quantity | quantitative_assay of the tyrosine tubulin positive axon tip. Data from three independent experiments were normalized to the solvent treated wt group. As can be seen from FIG. 2, parthenolide significantly increased the proportion of non-detyrosinated axonal tips of adult wild type neurons as measured in GSK3α ^{S / A} / GSK3β ^{S / A} neurons.

  This indicates that axonal growth of mature neurons caused by parthenolide is promoted through inhibition of microtubule detyrosination.

Determination of concentration dependence of the effect of parthenolide on sciatic nerve regeneration in vivo. Increasing concentrations of parthenolide of 0.01 μM, 0.05 μM, 0.1 μM, 1 μM, 10 μM and 100 μM were obtained in the wild immediately after sciatic nerve injury. The mice were administered within the sciatic nerve crush site of the mouse and the axon regeneration of the sciatic nerve section was evaluated to determine the concentration effect. Three days after sciatic nerve crush and treatment, the sciatic nerve was isolated and stained with an antibody against the regeneration-related protein SCG10. SCG10 positive axons were quantified 2 mm and 3 mm away from the lesion site labeled with carbon. In the vehicle and 0.05 μM parthenolide groups, 5 animals were used per group, and 2 animals each were used at other concentrations. Five sciatic nerve sections were analyzed per animal.

  FIG. 3 shows the quantification of axonal regeneration into the sciatic nerve three days after sciatic nerve crush after treatment with vehicle (veh) or various parthenolide concentrations. As can be seen from FIG. 3, a concentration in the range of 0.01 μM to 0.1 μM markedly increased the axonal regeneration of the damaged sciatic nerve 3 days after SNC, with higher concentrations above 10 μM Rather reduced regeneration.

4.1 In vivo intraparticular administration of parthenolide 4.1 Determination of in vivo sciatic nerve regeneration To determine whether intranerve administration of parthenolide promotes sciatic nerve regeneration in vivo, 50 nM parthenolide or solvent was used in the wild type The mice were administered into the sciatic nerve at the same time as the surgery, and the effect on regeneration was determined 3 days after SNC.

  FIGS. 4A and 4B show longitudinal sections of the sciatic nerve 3 days after SNC injected intranervely with vehicle (veh) or 50 nM parthenolide (par). Regenerating axons were immunohistochemically stained for SCG10 shown in FIG. 4A or pMAP1B shown in FIG. 4B. As can be seen from FIGS. 4A and 4B, parthenolide treatment allowed the axons to regenerate longer distances beyond the site of injury without increasing the level of pMAP1B in the axons. FIG. 4C shows the axon of animals treated with vehicle (veh) or parthenolide (par) as described above> 1.5 mm,> 2 mm,> 2.5 mm and> 3 mm away from the site of sciatic nerve injury. It is the figure which showed fixed_quantity | quantitative_assay. As can be seen in FIG. 4C, the administration of parthenolide significantly increased the number of axons at a distance of 2.5 mm beyond the lesion site by more than 6 times compared to the solvent-treated control. Axons at a distance of 3 mm beyond the injury site were observed 3 days after surgery only after treatment with parthenolide.

This finding indicates that a single injection was sufficient to significantly accelerate functional regeneration compared to solvent-treated controls. It is believed that repeated intraneuronal injections of TCP inhibitors or systemic administration of drugs can further accelerate axonal regeneration. Furthermore, the effect of parthenolide on promoting axonal regeneration was stronger than that of GSK3α ^{S / A} / GSK3β ^{S / A} knock-in animals, and was not accompanied by an increase in MAP1B phosphorylation.

4.2 Determining the reconstruction of the neuromuscular junction after parthenolide treatment To verify that regenerative axons have already begun to redistribute well to the target 4 days after parthenolide treatment, BTX of the long thumb extensor and Neurofilament staining was performed. Mice were sacrificed 4 days after treatment with SNC and 50 nM parthenolide (par) and the long thumb extensors were dissected and stained. FIG. 4D shows α-bungarotoxin (BTX) and neurofilament (NF) staining of a total tissue preparation of long thumb extensor muscles of mice treated with parthenolide (par) or vehicle (veh) 4 days after sciatic nerve crush. FIG. As shown in FIG. 4D, animals treated with parthenolide showed a neuromuscular junction but not a solvent-treated control.

4.3 Determination of functional recovery To test whether parthenolide administration also accelerated in vivo functional recovery, the regeneration results of adult wild type mice after treatment with 50 nM parthenolide after crushing the sciatic nerve were Were functionally evaluated using static scastic index (SSI) and von Frey tests.

  FIG. 4E shows adult wild type treated with parthenolide (par, n = 11) or vehicle (veh, n = 11) 1, 4, 7, 9, 12, 14 and 21 days after sciatic nerve crush (dpc). It is the figure which showed the fixed_quantity | quantitative_assay of the motor function recovery | decision determined by static scastic index (SSI) in a mouse | mouth. As can be seen in FIG. 4E, the animals treated with parthenolide showed significantly improved SSI scores compared to solvent-treated control animals already 4 days after injury, which was maintained over the entire observation period of 3 weeks.

  FIG. 4F shows the von Frey test in adult wild type mice treated with parthenolide (par, n = 11) or vehicle (veh, n = 11) 1, 4, 7, 12, 14 and 21 days after sciatic nerve crush. It is the figure which showed fixed_quantity | quantitative_assay of the sensory function recovery | decision determined by this. As can be seen in FIG. 4F, parthenolide treatment also accelerated sensory recovery. In the von Frey test, the first improvement can be detected on the 7th day from the injury, and still significant on the 12th and 14th days, the distance required for the axons to reach their respective targets for sensory recovery is more Reflects a long time.

  These data indicate that intranerve administration of parthenolide significantly promotes sciatic nerve regeneration in vivo and accelerates recovery of motor and sensory functions.

Determination of in vitro axonal growth following administration of parthenolide and kunicin Adult wild type (wt) and GSK3α / GSK3β as described in Gobrecht et al., Nature Communications 2014; 5: 4561. Dorsal root ganglion (DRG) neurons were isolated from the mice. DRG (T8-L6) was collected and 0.25% trypsin / EDTA (GE Healthcare, Chalfont St Giles, UK) in DMEM (Life Technologies, Carlsbad, CA) And 0.3% collagenase type IA (Sigma, St. Louis, MO), incubated for 45 minutes at 37 ° C. and 5% CO ₂ and mechanically dissociated. 10% fetal bovine serum (GE Healthcare), penicillin / streptomycin (500 U / ml; Merck Millipore, Billerica, Massachusetts, USA) and 5-fluoro-2'-desoxyuridine (100 nM; Sigma) Cells were resuspended in DMEM containing. In a 96-well plate (Nunc, Germany) coated with poly-D-lysine (PDL, 0.1 mg / ml; molecular weight <300,000 kDa; Sigma) and laminin (20 μg / ml; Sigma), Cells were cultured at 37 ° C. and 5% CO ₂ . Cells were treated with vehicle or 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, or 100 nM parthenolide (Sigma-Aldrich) or kunicin (Extrasynthesis sheath) and cultured for 2 days.

  After 48 hours incubation, fixation with 4% PFA (Sigma) and NeuN (1: 2,000; Abeam, ab177487, Cambridge, UK) and βIII-tubulin (1: 2,000; Covance) ), Axonal growth was determined by immunocytochemical staining with antibodies against Princeton, NJ). Imaging and quantification of total axon length and number of neurons per well was automatically performed by Pathway 855 microscope system (BD, Franklin Lakes, NJ, USA) and Attovision software to avoid quantification bias due to the experimenter. Mean axon length per neuron and neuron count per experimental group were normalized to the control group.

  FIG. 5 shows the quantification of axon growth in neuronal cultures. Data represent the mean ± SEM of at least 6 replicate wells and 3 independent experiments per experiment. Significance of differences between groups was assessed using the Holm-Sidak post hoc test after one-way or two-way analysis of variance (ANOVA). As can be seen from FIG. 5, parthenolide increased axon growth significantly and concentration-dependently. The strongest effect was measured at 1 nM and 5 nM, but concentrations above 100 nM reduced axonal growth in culture. This indicates that the effect of parthenolide was concentration dependent. General toxicity was not observed because there was no effect on cell number at all test concentrations. Compared to parthenolide, the derivative kunicin was similarly significant but less noticeable in promoting axon growth and was most effective at 0.5 nM.

Determination of in vivo sciatic nerve regeneration after intrathenal and systemic administration of parthenolide 6.1: Intraneuronal parthenolide administration As described in Example 4.1, 1.25 pg, 6.25 pg, 12.5 pg, 125 pg, Parthenolide doses of 1,250 pg and 12,500 pg were administered simultaneously with surgery at the site of sciatic nerve crush in wild type mice and the effect on regeneration was determined after 3 days.

  6A, B, and C show 3 days after performing a single intraneuronal (in) injection of sciatic nerve crush (SNC) and vehicle (top) or parthenolide (6.25 pg par; medium). It is the figure which showed the typical longitudinal section of the sciatic nerve. 6B and 6C are enlarged views of each region shown in A. FIG. As can be seen from FIGS. 6A, B and C, only a few axon profiles were detected about 2.5 mm away from the lesion of the solvent-injected animal (B), but significant after intranerve (C) parthenolide injection. There were many regenerative axons. FIG. 6E shows axon quantification of longitudinal sections 1.5 mm, 2 mm, 2.5 mm and 3 mm away from the sciatic nerve injury site in mice injected intraventrally with vehicle (veh) or parthenolide. It is. As can be seen from FIG. 6E, doses ranging from 1.25 to 12.5 pg administered intraneurally significantly increased axonal regeneration 3 days after SNC. The strongest growth promotion was determined at 6.25 pg and 12.5 pg doses, which increased the number of axons at a distance of 2.5 mm from the lesion site by more than 3 times compared to the solvent-treated controls. .

6.2: Systemic administration of parthenolide In order to test whether systemic parthenolide administration can promote sciatic nerve regeneration, 20 ng / kg, 200 ng / kg, 2 μg / kg and 20 μg / kg parthenolide doses were administered. Intraperitoneal injection was performed after nerve injury.

  6A and D show longitudinal sections of the sciatic nerve 3 days after sciatic nerve crush (SNC) and intraperitoneal (ip) parthenolide injection (200 ng / kg; bottom). 6D is an enlarged view of each region after intraperitoneal (ip) parthenolide injection shown in A. FIG. As can be seen in FIGS. 6A and D, there were significantly more regenerating axons after intraperitoneal (D) partenolide injection. FIG. 6F is a diagram showing the quantification of axons of longitudinal sections 1.5 mm, 2 mm, 2.5 mm and 3 mm ahead from the damaged site of the sciatic nerve injected intraperitoneally with solvent (veh) or parthenolide. As can be seen from FIG. 6F, a single injection of 200 ng / kg significantly increased the number of regenerating axons at 2.5 mm by about 2.5 times, which is somewhat evident compared to intraneural parthenolide administration. Is inferior. Higher test doses did not significantly affect sciatic nerve regeneration.

  These data indicate that systemic administration of parthenolide also significantly promotes sciatic nerve regeneration in vivo and accelerates recovery of motor and sensory functions.

Determination of the effect of parthenolide on cells of the central nervous system To test whether parthenolide is effective on cells of the central nervous system, the neurite extension of adult retinal ganglion cells was determined. For this purpose, adult mouse retinas were dissociated and cultured for 4 days in the presence of solvent (−), lithium (li) or parthenolide (Par, 1 nM). Cells were fixed and stained for βIII-tubulin to determine the neurite length for each retinal ganglion cell. Quantification of neurite outgrowth of retinal ganglion cells after treatment with 1 nM parthenolide (par), lithium (li), or both is shown in FIG. As can be seen from FIG. 7, parthenolide significantly and significantly promoted neurite growth compared to untreated controls. Lithium did not have a significant effect, but further enhanced the beneficial effect of parthenolide.

  These data indicate that parthenolide can promote nerve regeneration not only in peripheral nerves such as the sciatic nerve but also in central neurons. This finding suggests that parthenolide may also be useful for promoting CNS regeneration, such as after optic nerve or spinal cord injury.

These data together provide a novel therapeutic approach for promoting nerve regeneration and for the treatment of nerve damage. In vivo pharmacological TCP inhibition with parthenolide inhibits detyrosination of damaged nerve growth cone microtubules, a novel and clinically feasible method to accelerate axonal regeneration and improve functional recovery It has been shown to provide a simple approach.

Claims (15)

  A compound that reduces de-tyrosination of axonal tip microtubules selected from the group consisting of a tubulin carboxypeptidase inhibitor and a tubulin tyrosine ligase activator and combinations thereof for the treatment of axonal disorders.   The tubulin carboxypeptidase inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. The compound of claim 1.   2. A compound according to claim 1, wherein the axonal disorder is damage to the peripheral or central nervous system, in particular central projection of the sciatic or optic nerve or spinal cord.   2. The compound of claim 1, wherein the axonal disorder is associated with peripheral neuropathy, glaucoma, stroke or multiple sclerosis.   2. A compound according to claim 1, wherein the axonal disorder is associated with denervation of the damaged or transplanted cornea, or associated with a lesioned optic or trigeminal nerve, in particular an axotomy fiber of the eye branch.   A compound that reduces detyrosination of microtubules at the tip of axons selected from the group consisting of tubulin carboxypeptidase inhibitors and tubulin tyrosine ligase activators and combinations thereof for the treatment of axonal disorders A pharmaceutical composition comprising.   The tubulin carboxypeptidase inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. The pharmaceutical composition according to claim 7.   8. The composition of claim 7, wherein the composition is formulated for topical administration such as intranerve administration or perineural administration, or systemic administration such as intraperitoneal, intravenous, subcutaneous or oral administration. Pharmaceutical composition.   The pharmaceutical composition according to claim 7, wherein the composition is formulated for intraocular administration, in particular as an eye drop.   Detyrosineation of microtubules at the tip of axons selected from the group consisting of tubulin carboxypeptidase inhibitors and tubulin tyrosine ligase activators and combinations thereof for the manufacture of drugs for the treatment of axonal disorders Use of reducing compounds.   The tubulin carboxypeptidase inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. Use according to claim 10.   Said axonal disorder is damage of the peripheral or central nervous system, in particular central projection of the sciatic or optic nerve or spinal cord, or is related to or involved in denervation of the damaged or transplanted cornea 11. Use according to claim 10, in relation to the optic nerve or trigeminal nerve that has developed, in particular to the axotomy fiber of its eye branch.   A method of treating an axonal disorder, wherein the therapy reduces adenosine tip microtubule detyrosination selected from the group consisting of a tubulin carboxypeptidase inhibitor and a tubulin tyrosine ligase activator and combinations thereof Administering a top effective amount of the compound to the subject.   The tubulin carboxypeptidase inhibitor is parthenolide or its racemate, enantiomer, stereoisomer, solvate, hydrate, pharmaceutically acceptable salt, ester and / or derivative or structural analog. The method of claim 13. Said axonal disorder is damage of the peripheral or central nervous system, in particular central projection of the sciatic or optic nerve or spinal cord, or is related to or involved in denervation of the damaged or transplanted cornea 14. The method according to claim 13, wherein the method is associated with the optic nerve or trigeminal nerve that has developed, especially the axotomy fiber of the eye branch.